We will undertake experiments designed to fractionate, purify and characterize the enzyme glutathione peroxidase and its selenium prosthetic group in order to define the unique chemical and biochemical properties of selenium in this enzyme. Methods for purification of glutathione peroxidase and its prosthetic group include enzymatic activity assay, gel filtration, ion exchange, thin layer and paper chromatography, polyacrylamide gel electrophoresis and Se75 radioisotope methodology. Further chemical characterization of the selenium prosthetic group will be accomplished using the techniques of gas chromatography alone and in combination with mass spectrometer-computer system, I.R., U.V. and N.M.R. spectroscopy. Data obtained on the unique enzymatic properties of glutathione peroxidase derived via substrate kinetics, spectral characterization and enzyme inhibition studies will be related to the chemical structure of the selenium prosthetic group to develop and define the enzymatic mechanism by which hydroperoxides are reduced and the manner by which selenium is incorporated nutritionally as a prosthetic function. Using in vitro systems containing peroxides or peroxidizing polyunsaturated lipids, peroxide labile enzyme and the protective enzyme glutathione peroxidase, we will investigate the mechanism by which this enzyme prtects biomolecules against peroxidative damage. By the measurement of subcellular and tissue distribution of glutathione peroxidase and other biochemically related components, we will define the in vivo glutathione peroxidase protection system. Using experiments in which animals are exposed to heavy metals, atmospheric oxidant and lipid peroxides, we plan to determine the response and capacity of this system in protecting against these environmentally relevant stresses.